Stock No: 000308
Protocol 35291: Probe Assay - Ednrb<s>-Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant=  66 bp

Wild Type = 92 bp

Sequence

Wt Sequence:
ttcaggggtggggaagaaataatgtaaacagaAACCATActgattagttctgaggatagaaagcaaacaaacaaacaaaaagggatatagctggggaccaacatc
 
Mutant Sequence:
GGGAGACCCTGATCAGAGGGTTGTTGCCAATGCAATTGAGTTCAGGGGTGGGGAAGAAATAATGTAAACAGAAActGTCAGAGACCATCCCGTGAGAAAGTGAGCCCTTC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35115 GAA GGG CTC ACT TTC TCA CG Mutant Reverse A
45701 GGG GTG GGG AAG AAA TAA TG Common A
45702 CCC CAG CTA TAT CCC TTT TTG T Wild type Reverse A
45703 Fluorophore-1 TAA ACA GAA ACC ATA CTG ATT AGT TCT GA Quencher-1 WT Probe
45704 Fluorophore-2 TAA ACA GAA ACT GTC AGA GAC CAT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35115 0.40 uM
45701 0.40 uM
45702 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.